DNA encoding a novel human anion channel

ABSTRACT

The present invention provides a novel anion channel (NANCH) and polynucleotides which identify and encode NANCH. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding NANCH and a method for producing NANCH. The invention also provides for agonists, antibodies, or antagonists specifically binding NANCH, and their use, in the prevention and treatment of diseases associated with expression of NANCH. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding NANCH for the treatment of diseases associated with the expression of NANCH. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding NANCH.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of anovel human anion channel and to the use of these sequences in thediagnosis, prevention, and treatment of cancer and developmentaldisorders.

BACKGROUND OF THE INVENTION

Chloride channels are found in the plasma membranes of virtually everycell in the body. Chloride channels mediate a variety of cellularfunctions including regulation of membrane potentials and absorption andsecretion of ion across epithelial membranes. When present inintracellular membranes of the Golgi apparatus and endocytic vesicles,chloride channels also regulate organelle pH (cf. Greger, R. (1988)Annu. Rev. Physiol. 50:111-122). Electrophysiological andpharmacological measurenments including ion conductance, current-voltagerelationships, and sensitivity to modulators suggest that differentchloride channels exist in muscles, neurons, fibroblasts, epithelialcells, and lymphocytes.

Several chloride channels have been cloned Trom mammalian tissues andcell lines. The sequences of these proteins are diverse and include anicotinic acetylcholine receptor homolog, the cystic fibrosistransmembrane conductance regulator, and the highly acidic p64 proteinwith no significant homology to other proteins (Bernard, E. A. et al.(1987) Trends Neurosci. 16:502-509; Riordan, J. R. et al. (1989) Science245:1066-1073; Landry, D. et al. (1993) J. Biol. Chem. 268:14948-14955).Many of the channels have sites for phosphorylation by one or moreprotein kinases including protein kinase A, protein kinase C, tyrosinekinase, and casein kinase II which regulate chloride channel activity inepithelial cells.

The p64 protein was originally identified in bovine kidney cortexmembranes by its affinity for indanyloxyacetic acid, a known inhibitorof epithelial chloride channels (Landry et al. (1989) Science244:1469-1472). Antibodies raised against the isolated p64 protein candeplete solubilized kidney membranes of all detectable chloride channelactivity. Thus, p64 is likely to be a functional component of the kidneychloride channel (Redhead, R. C. et al. (1992) Proc. Natl. Acad. Sci.89:3716-3720).

Northern blot analyses using the p64 clone as a probe detect relatedmRNAs, ranging in size from ˜2 kb to ˜6.5 kb, in bovine kidney cortex,kidney medulla, liver, adrenal glands, brain, skeletal muscle, andheart. Most of these tissues have multiple transcripts that are capableof hybridizing to this probe. The diversity and relative abundance ofthese transcripts is tissue-specific, and this suggests that the p64transcripts are alternatively spliced and/or that a family of relatedgenes are expressed (Landry et al., supra).

The sequence of p64 predicts an acidic, integral membrane protein whichspans the membrane at least twice and has the amino terminus on thecytoplasmic side. The protein has potential sites for phosphorylation byprotein kinase C, tyrosine kinase, and casein kinase II, and a singlesite for N-inked glycosylation at Asp²³⁵.

The discovery of polynucleotides encoding a novel human anion channel,and the molecules themselves, provides a means to investigate theregulation of membrane potentials, intracellular pH, cell volume, signaltransduction, and tansepithelial ion transport in tissues containingabsorptive or secretory epithelia under normal and disease conditions.Discovery of a novel anion channel satisfies a need in the art byproviding new compositions useful in diagnosing and treating cancer anddevelopmental disorders.

SUMMARY OF THE INVENTION

The present invention features a novel human anion channel proteinhereinafter designated NANCH and characterized as having similarity tobovine p64 chloride channel and human P64CLCP.

Accordingly, the invention features a substantially purified NANCHhaving the amino acid sequence shown in SEQ ID NO:1.

One aspect of the invention features isolated and substantially purifiedpolynucleotides that encode NANCH. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2.

The invention also relates to a polynucleotide sequence comprising thecomplement of SEQ ID NO:2 or variants thereof. In addition, theinvention features polynucleotide sequences which hybridize understringent conditions to SEQ ID NO:2.

The invention additionally features nucleic acid sequences encodingpolypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments,portions or antisense molecules thereof, and expression vectors and hostcells comprising polynucleotides that encode NANCH. The presentinvention also features antibodies which bind specifically to NANCH, andpharmaceutical compositions comprising substantially purified NANCH. Theinvention also features the use of agonists and antagonists of NANCH.The invention also features methods for treating disorders which areassociated with NANCH, and for detecting a polynucleotide which encodesNANCH.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C shows the amino acid sequence (SEQ ID NO:1) and nucleic acidsequence (SEQ ID NO:2) of NANCH. The alignment was produced usingMacDNASIS PRO™ software (Hitachi Software Engineering Co., Ltd., SanBruno, Calif.).

FIGS. 2A-2C shows the amino acid sequence alignments among NANCH (SEQ IDNO:1), bovine p64 chloride channel (GI 289404; SEQ ID NO:3), and humanP64CLCP (GI 895845; SEQ ID NO:4). The alignment was produced using themultisequence alignment program of DNASTART™ software (DNASTAR Inc,Madison Wis.).

FIGS. 3A and 3B show the hydrophobicity plots (produced using theprotein analysis program of DNASTAR software) for NANCH, SEQ ID NO:1,and bovine p64 chloride channel, SEQ ID NO:3, respectively; the positiveX axis reflects amino acid position, and the negative Y axis,hydrophobicity.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

DEFINITIONS

"Nucleic acid sequence" as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomnic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand. Similarly,"amino acid sequence" as used herein refers to an oligopeptide,pepticle, polypeptide, or protein sequence, and fragments or portionsthereof, and to naturally occurring or synthetic molecules.

Where "amino acid sequence" is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, "amino acidsequence" and like terms, such as "polypeptide" or "protein" are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

"Peptide nucleic acid", as used herein, refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop tnanscript elongation by binding totheir complementary strand of nucleic acid Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

NANCH, as used herein, refers to the amino acid sequences ofsubstantially purified NANCH obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, or which has been extendedusing XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3'direction and resequenced, or which has been assembled from tieoverlapping sequences of more than one Incyte clone using the GELVIEW™Fragment Assembly system (GCG, Maidison, Wis.), or which has been bothextended and assembled.

A "variant" of NANCH, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

A "deletion", as used herein, refers to a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent.

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to thenaturally occurring molecule.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic NANCH, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The term "agonist", as used herein, refers to a molecule which, whenbound to NANCH, causes a change in NANCH which modulates the activity ofNANCH. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to NANCH.

The terms "antagonist" or "inhibitor", as used herein, refer to amolecule which, when bound to NANCH, blocks or modulates the biologicalor immunological activity of NANCH. Antagonists and inhibitors mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to NANCH.

The term "modulate", as used herein, refers to a change or an alterationin the biological activity of NANCH. Modulation may be an increase or adecrease in protein activity, a change in binding characteristics, orany other change in the biological, functional or immunologicalproperties of NANCH.

The term "mimetic", as used herein, refers to a molecule, the structureof which is developed from knowledge of the structure of NANCH orportions thereof and, as such, able to effect some or all of the actionsof bovine p44ike molecules.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding NANCH or the encoded NANCH.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

"Amplification" as used herein refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "hybridization", as used herein, refers to any process by whicha strand olf nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen binds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparalel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, for the sequence"A-G-T" binds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term"substantially homologous." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific bindirng, the probe will not hybridize tothe second non-complementary target sequence.

As known in the art, numerous equivalent conditions may be employed tocomprise either low or high stringency conditions. Factors such as thelength and nature (DNA, RNA, base composition) of the sequence, natureof the target (DNA, RNA, base composition, presence in solution orimmobilization, etc.), and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfateand/or polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency different from, but equivalent to, the above listedconditions.

The term "stringent conditions", as used herein, is the "stringency"which occurs within a range from about Tm-5° C. (5° C. below the meltingtemperature (Tm) of the probe) to about 20° C. to 25° C. below Tm. Aswill be understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences.

The term "antisense", as used herein, refers to nucleotide sequenceswhich are complementary to a specific DNA or RNA sequence. The term"antisense strand" is used in reference to a nucleic acid strand that iscomplementary to the "sense" strand. Antisense molecules may be producedby any method, including synthesis by ligating the gene(s) of interestin a reverse orientation to a viral promoter which permits the synthesisof a complementary strand. Once introduced into a cell, this transcribedstrand combines with natural sequences produced by the cell to formduplexes. These duplexes then block either the further transcription ortranslation. In this manner, mutant phenotypes may be generated. Thedesignation "negative" is sometimes used in reference to the antisensestrand, and "positive" is sometimes used in reference to the sensestrand.

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO:1" encompassestie full-length human NANCH and fragments thereof.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. Tlhe method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosorae. They alsoinclude cells which transiently express the inserted DNA or RNA forlimited periods of time.

The term "antigenic determinant", as used herein, refers to that portionof a molecule that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The terms "specific binding" or "specifically binding", as used herein,in reference to the interaction of an antibody and a protein or peptide,mean that the interaction is dependent upon the presence of a particularstructure (i.e., the antigenic determinant or epitope) on the protein;in other words, the antibody is recognizing and binding to a specificprotein structure rather than to proteins in general. For example, if anantibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding NANCH orfragments thereof may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2 by northern analysis is indicative of thepresence of mRNA encoding NANCH in a sample and thereby correlates withexpression of the transcript from the polynucleotide encoding theprotein.

"Alterations" in the polynucleotide of SEQ ID NO:2, as used herein,comprise any alteration in the sequence of polynucleotides encodingNANCH including deletions, insertions, and point mutations that may bedetected using hybridization assays. Included within this definition isthe detection of alterations to the genomic DNA sequence which encodesNANCH (e.g., by alterations in the pattern of restriction fragmentlength polymorphisms capable of hybridizing to SEQ ID NO:2), theinability of a selected fragrent of SEQ ID NO:2 to hybridize to a sampleof genomic DNA (e.g., using allele-specific oligonucleotide probes), andimproper or unexpected hybridization, such as hybridization to a locusother than the normal chromosomal locus for the polynucleotide sequenceencoding NANCH (e.g., using fluorescent in situ hybridization (FISH) tometaphase chromosomes spreads).

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fa, F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind NANCHpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

THE INVENTION

The invention is based on the discovery of a novel human anion channel(NANCH), the polynucleotides encoding NANCH, and the use of thesecompositions for the diagnosis, prevention, or treatment of cancer anddevelopmental disorders.

The nucleic acid sequence encoding the human NANCH of the presentinvention was first identified in Incyte Clone 259592 from an hNT2neuronal cell line cDNA library (HNT2RAT01) through a computer-generatedsearch for amino acid sequence alignments. A consensus sequence, SEQ IDNO:2, was derived from the following overlapping and/or extended nucleicacid sequences: Incyte Clones 040177 (TBLYNOT01); 259592 (HNT2RAT01);311528 (LUNGNOT02); 760901 (BRAITUT02); 1522436 (BLADTUT04); and 2134172(ENDCNOT01).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A and 1B.NANCH is 253 amino acids in length and has potential transmembranedomains at residues 19-54 (which is long enough to span the membranetwice) and residues 187-201, and a potential β-sheet-formingtransmembrane domain at residues 116-129. NANCH contains consensussequences for phosphorylation by protein kinase C and casein kinase IIat S²³⁶, tyrosine kinase at Y²⁴⁴, and cAMP-dependent protein kinase atT²⁵². These potential phosphorylation sites are all located near theC-terminus of NANCH. NANCH has chemnical and structural homology withbovine p64 chloride channel (GI 289404; SEQ ID NO:3) and human P64CLCP(GI 895845; SEQ ID NO:4). This suggests that NANCH is involved in theregulation of transepithelial ion transport in tissues containingabsorptive or secretory epithelia.

In the region of homology, NANCH shares 76% and 65% identity with GI289404 and GI 895845, respectively (FIGS. 2A and 2B). GI 289404 isconsiderably longer than NANCH or GI 895845, and its first 200 aminoacid residues have no significant counterparts in either NANCH or GI895845. This may reflect species differences, alternative splicing,and/or transcription from different members of the gene family. Asillustrated by FIGS. 3A and 3B, NANCH and GI 289404 have similarhydrophobicity plots in the region of homology. Northern analysisreveals the expression of the NANCH sequence in libraries derived fromvascular endothelial cells and left ventricle of the heart, and intissues involved in secretion and adsorption including stomach, lung,kidney, tongue, colon, large intestine, pancreas, adrenal gland,thyroid, bladder, and pancreas. Sixteen of the libraries (55%) arederived from tumors and fetal tissues, suggesting that NANCH isassociated with regulation of cell growth.

The invention also encompasses NANCH variants. A preferred NANCH variantis one having at least 80%, and more preferably 90%, amino acid sequenceidentity to the NANCH amino acid sequence (SEQ ID NO: 1). A mostpreferred NANCH variant is one having at least 95% amino acid sequenceidentity to SEQ ID NO:1.

The invention also encompasses polynucleotides which encode NANCH.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of NANCH can be used to generate recombinant molecules whichexpress NANCH. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A and 1B.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding NANCH, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring NANCH, and all such variations are to beconsidered as being specifically disclosed.

Although nucleotide sequences which encode NANCH and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring NANCH under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding NANCH or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occult in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding NANCH and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or portionsthereof., which encode NANCH and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art at the time of the filing ofthis application. Moreover, synthetic chemistry may be used to introducemutations into a sequence encoding NANCH or any portion thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

Altered nucleic acid sequences encoding NANCH which are encompassed bythe invention include deletions, insertions, or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent NANCH. The encoded protein may alsocontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentNANCH. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of NANCH is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

Also included within the scope of the present invention are alleles ofthe genes encoding NANCH. As used herein, an "allele" or "allelicsequence" is an alternative form of the gene which may result from atleast one mutation in the nucleic acid sequence. Alleles may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given gene may have none, one, or many allelic forms.Common mutational changes which give rise to alleles are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any embodiments of the invention. Themethods may employ such enzymes as the Klenow fragment of DNA polymeraseI, Sequenase® (US Biochemical Corp, Cleveland, Ohio), Taq polymerase(Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), orcombinations of recombinant polyinerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Gibco BRL(Gaithersburg, Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), PeltierThermal Cycler (PTC200; M.J. Research, Watertown, Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

The nucleic acid sequences encoding NANCH may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 Primer Analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymnes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationaxid used as a PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and litigations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5' and 3'non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devisecamera Output/light intensity may be converted to electrical signalusing appropriate software (e.g. Genotype™ and Sequence Navigator™,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlledCapillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode NANCH, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of NANCH in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressNANCH.

As will be understood by those of skill in the art, it may beadvantageous to produce NANCH-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter NANCHencoding sequences for a variety of reasons, including but not limitedto, alterations which modify the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding NANCH may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of NANCH activity, it may be useful toencode a chimeric NANCH protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the NANCH encoding sequence and theheterologous protein sequence, so that NANCH may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding NANCH may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nuc. Acids Res. Symp. Ser. 215-223, Horn,T. et al. (1980) Nuc. Acids Res. Symp. Ser. 225-232). Alternatively, theprotein itself may be produced using chemical methods to synthesize theamino acid sequence of NANCH, or a portion thereof. For example, peptidesynthesis can be performed using various solid-phase techniques(Roberge, J. Y. et al. (1995) Science 269:202-204) and automatedsynthesis may lee achieved, for example, using the ABI 431A PeptideSynthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton T.(1983) Proteins, Structures and Molecular Principles, W.H. Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, T., supra). Additionally, the aminoacid sequence of NANCH, or any part thereof, may be altered duringdirect synthesis and/or combined using chemical methods with sequencesfrom other proteins, or any part thereof, to produce a variantpolypeptide.

In order to express a biologically active NANCH, the nucleotidesequences encoding NANCH or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding NANCH andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques wedescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology. John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding NANCH. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL) and Vie like may be used.The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO: and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding NANCH,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for NANCH. For example, when largequantities of NANCH are needed for the induction of antibodies, vectorswhich direct high level expression of fusion protein, that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding NANCH may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding NANCH may be driven by any of a number of promoters.For example, viral apromoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamitsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced into)plant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196.

An insect system may also be used to express NANCH. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding NANCH may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of NANCH will render the polyhedrin gene inactive and producerecombinant. virus lacking coat protein. The recombinant viruses maythen be used to infect, for example., S. frugiperda cells orTrichopltisia larvae in which NANCH may be expressed (Engelhard, E. K.et al. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding NANCH may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing NANCH in infected host cells (Logan, J. and T.Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding NANCH. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding NANCH, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG; initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and ac) lation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressNANCH may be transformed using expression vectors which may containviral origins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltrrnsferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β-glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding NANCH isinserted within a marker gene sequence, recombinant cells containingsequences encoding NANCH can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding NANCH under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding NANCH and express NANCH may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding NANCH can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orportions or fragments of polynucleotides encoding NANCH. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding NANCH to detect transformantscontaining DNA or RNA encoding NANCH. As used herein "oligonucleotides"or "oligomers" refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofNANCH, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson NANCH is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding NANCH includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding NANCH, orany portions thereof may be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio).Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with nucleotide sequences encoding NANCH may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeNANCH may be designed to contain signal sequences which direct secretionof NANCH through a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may be used to join sequences encoding NANCHto nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and NANCH may be used to facilitate purification.One such expression vector provides for expression of a fusion proteincontaining NANCH and a nucleic acid encoding 6 histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography as described in Porath, J. et al. (1992, Prot.Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides ameans for purifying NANCH from the fusion protein. A discussion ofvectors which contain fusion proteins is provided in Kroll, D. J. et al.(1993; DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of NANCH may beproduced by direct peptide synthesis using solid-phase techniquesMerrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Various fragments of NANCH may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

THERAPEUTICS

Chemical and structural homology exists among NANCH, bovine p64, andhuman P64CLCP. This homology together with its northern analysissuggests that NANCH has a role in development. Accordingly, lack ofNANCH may be implicated in developmental disorders.

Therefore, in one embodiment, NANCH or a fragment or derivative thereofmay be administered to a subject to treat developmental disorders. Suchdisorders may include, but are not limited to, incomplete development ofany organ, including the heart, lungs, kidneys or liver in neonates, aswell as congenital defects.

In another embodiment, a vector capable of expressing NANCH, or afragment or a derivative thereof, may also be administered to a subjectto treat the developmental disorders listed above.

NANCH also appears to have a role in the development of cancer.Therefore, in one embodiment, antagonists or inhibitors of NANCH many beadministered to a subject to treat or prevent any type of cancerincluding, but not limited to, those of the heart, stomach, lung, skin,kidney, tongue, colon, large intestine, liver, pancreas, adrenal gland,thyroid, bladder, and pancreas. In one aspect, antibodies which arespecific for NANCH may be used directly as an antagonist, or indirectlyas a targeting or delivery mechanism for bringing a pharmaceutical agentto cells or tissue which express NANCH.

In another embodiment, a vector expressing antisense of thepolynucleotide encoding NANCH may be administered to a subject to treator prevent the types of cancer including, but not limited to, thoselisted above.

In other embodiments, any of the therapeutic proteins, antagonists,antibodies, agonists, antisense sequences or vectors described above maybe administered in combination with other appropriate therapeuticagents. Selection of the appropriate agents for use in combinationtherapy may be made by one of ordinary skill in the art, according toconventional pharmaceutical principles. The combination of therapeuticagents may act synergistically to effect the treatment or prevention ofthe various disorders described above. Using this approach, one may beable to achieve therapeutic efficacy with lower dosages of each agent,thus reducing the potential for adverse side effects.

Antagonists or inhibitors of NANCH may be produced using methods whichare generally known in the art. In particular, purified NANCH may beused to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind NANCH. Antibodies toNANCH may be generated using methods that are well known, in the art.Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies, (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith NANCH or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the peptides, fragments, or oligopeptides used toinduce antibodies to NANCH have an amino acid sequence consisting of atleast five amino acids, and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of NANCH amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

Monoclonal antibodies to NANCH may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-12; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberper, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceNANCH-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for NANCH mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragmets which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between NANCH and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering NANCH epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingNANCH, or any fragment thereof, or antisense molecules, may be used fortherapeutic purposes. In one aspect, antisense to the polynucleotideencoding NANCH may be used in situations in which it would be desirableto block the transcription of the mRNA. In particular, cells may betransformed with sequences complementary to polynucleotides encodingNANCH. Thus, antisense molecules may be used to modulate NANCH activity,or to achieve regulation of gene function. Such technology is now wellknown in the art, and sense or antisense oligomers or larger fragments,can be designed from various locations along the coding or controlregions of sequences encoding NANCH.

Expression vectors derived from retro viruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingNANCH. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

Genes encoding NANCH can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes NANCH. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA, or PNA, to the control regionsof the gene encoding NANCH, i.e., the promoters, enhancers, and introns.Oligonucleotides derived from the transcription initiation site, e.g.,between positions -10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using, "triple helix" base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The antisense molecules may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding NANCH.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of nucleic acid molecules.These include techniques for chemically synthesizing oligonucleotidessuch as solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences encoding NANCH. Such DNA sequences may be incorporatedinto a wide variety of vectors with suitable RNA polymerase promoterssuch as T7 or SP6. Alternatively, these cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flaking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl phosphodiester linkageswithin the backbone of the molecule. This concept is inherent in theproduction of PNAs and can be extended in all of these molecules by theinclusion of nontraditional bases such as inosine, queosine, andwybutosine, as well as acetyl-, methyl-, thio-, and similarly modifiedforms of adenine, cytidine, guanine, thymine, and uridine which are notas easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by, liposome injections maybe achieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of NANCH, antibodies toNANCH, mimetics, agonists, antagonists, or inhibitors of NANCH. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be usect orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric.sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of NANCH, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example NANCH or fragments thereof, antibodies of NANCH,agonists, antagonists or inhibitors of NANCH, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose rntio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different Formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind NANCH may beused for the diagnosis of conditions or diseases characterized byexpression of NANCH, or in assays to monitor patients being treated withNANCH, agonists, antagonists or inhibitors. The antibodies usefull fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for NANCH includemethods which utilize the antibody and a label to detect NANCH in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuringNANCH are known in the art and provide a basis for diagnosing altered orabnormal levels of NANCH expression. Normal or standard values for NANCHexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toNANCH under conditions suitable for complex formation The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric, means. Quantities of NANCH expressed insubject, control and disease, samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingNANCH may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, antisense RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofNANCH may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression ofNANCH, and to monitor regulation of NANCH levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding NANCH or closely related molecules, may be used to identifynucleic acid sequences which encode NANCH. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding NANCH, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe NANCH encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring NANCH.

Means for producing specific hybridization probes for DNAs encodingNANCH include the cloning of nucleic acid sequences encoding NANCH orNANCH derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding NANCH may be used for the diagnosis ofconditions or diseases which are associated with expression of NANCH.Examples of such conditions or diseases include cancers of the heart,stomach, lung, skin, kidney, tongue, colon, large intestine, liver,pancreas, adrenal gland, thyroid, bladder, and pancreas. Thepolynucleotide sequences encoding NANCH may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dip stick, pin, ELISA or chip assays utilizingfluids or tissues from patient biopsies to detect altered NANCHexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding NANCH may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingNANCH may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding NANCH in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of NANCH, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes NANCH, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained front samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding NANCH may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5'→3') and another with antisense(3'←5'), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitative of closelyrelated DNA or RNA sequences.

Methods which may also be used to quantitate the expression of NANCHinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).The speed of quantitative of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or colorimetricresponse gives rapid quantitative.

In another embodiment of the invention, the nucleic acid sequences whichencode NANCH may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques. Such techniquesinclude FISH, FACS, or artificial chromosome constructions, such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

FISH (as described in Verma et al. (1988) Human Chromosomes: A ManualBasic Techniques, Pergamon Press, New York, N.Y.) may be correlated withother physical chromosome mapping techniques and genetic map data.Examples of genetic map data can be found in the 1994 Genome Issue ofScience (265:1981f). Correlation between the location of the geneencoding NANCH on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated marker even if the number or arm of a particular humanchromosome is not known. New sequences can be assigned to chromosomalarms, or parts thereof, by physical mapping. This provides valuableinformation to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, NANCH, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in an. of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on at cell surface, orlocated intracellularly. The formation of binding complexes, betweenNANCH and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to NANCH large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with NANCH, or fragments thereof, and washed. Bound NANCH isthen detected by methods well known in the art. Purified NANCH can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding NANCH specificallycompete with a test compound for binding NANCH. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with NANCH.

In additional embodiments, the nucleotide sequences which encode NANCHmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

IHNT2RAT01 cDNA Library Construction

The hNT2 cell line exhibits characteristics of a committed neuronalprecursor cell which is still at an early stage of development. The hNT2cell line can be induced by retinoic acid (RA) to differentiate, asdescribed in Andrews, P. W. (1984; Dev. Biol. 103:285-293). For purposesof this invention, hNT2 cells were induced with RA by suspending thecells in Dulbecco's modified Eagle's medium (DMEM) including 10% fetalbovine serum plus penicillin/streptomycin and treating with 10 μM RA for24 hours. The cells were harvested immediately thereafter.

The HNT2RAT01 cDNA library prepared from this treated cell line wasprepared by Stratagene (Cat. No. 937231). The cDNA library wasconstructed by essentially the following procedure. cDNAs were primedusing oligo d(T) and size fractionated to isolate fragments of 500 bpand larger. Synthetic adapter oligonucleotides were ligated onto thecDNA molecules enabling them to be inserted into the Uni-ZAPTh™ vectorsystem (Stratagene). The quality of the cDNA library was screened usingDNA probes, and the pBluescript phagemid (Stratagene) was then excised.Subsequently, the custom-constructed library phage particles wereinfected into E. coli host strain XL1-Blue® (Stratagene).

II Isolation and Sequencing of cDNA Clones

Plasmid or phagemid DNA was released from cells and purified using theMiniprep Kit (Cat. No. 77468; Advanced Genetic Technologies Corporation,Gaithersburg Md.). This kit consists of a 96 well block with reagentsfor 960 purifications. The recommended protocol was employed except forthe following changes: 1) the 96 wells were each filled with only 1 mlof sterile Terrific Broth (Cat. No. 22711, LIFE TECHNOLOGIES™,Gaithersburg Md.) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2)the bacteria were cultured for 24 hours after the wells were inoculatedand then lysed with 60 μl of lysis buffer; 3) a centrifugation stepemploying the Beckman GS-6R at 2900 rpm for 5 min was performed beforethe contents of the block were added to the primary filter plate; and 4)the optional step of adding isopropanol to TRIS buffer was not routinelyperformed. After the last step in the protocol, samples were transferredto a Beckman 96-well block for storage.

Alternative methods of purifying plasmid DNA include the use of MAGICMINIPREPS™ DNA Purification System (Cat. No. 7100, Promega) orQIAwell™-8 Plasmid, QIAwell PLUS DNA and QIAwell ULTRA DNA PurificationSystems (Qiagen, Inc.).

The cDNAs were sequenced by the method of Sanger F. and A. R. Coulson(1975; J. Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200(Hamilton, Reno Nev.) in combination with four Peltier Thermal Cyclers(PTC200 from MJ Research, Watertown Mass.) and Applied Biosystems 377 or373 DNA Sequencing Systems (Perkin Elmer) and reading frame wasdetermined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

Each cDNA was compared to sequences in GenBank using a search algorithmdeveloped by Applied Biosystems and incorporated into the INHERIT™ 670sequence analysis system. In this algorithm, Pattern SpecificationLanguage (TRW Inc, Los Angeles, Calif.) was used to determine regions ofhomology. The three parameters that determine how the sequencecomparisons run were window size, window offset, and error tolerance.Using a combination of these three parameters, the DNA database wassearched for sequences containing regions of homology to the querysequence, and the appropriate sequences were scored with an initialvalue. Subsequently, these homologous regions were examined using dotmatrix homology plots to distinguish regions of homology from chancematches. Smith-Waterman alignments were used to display the results ofthe homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT-670 sequence analysis system using the methods similar to thoseused in DNA sequence homologies. Pattern Specification Language andparameter windows were used to search protein databases for sequencescontaining regions of homology which were scored with an initial value.Dot-matrix homology plots were exanined to distinguish regions ofsignificant homology from chance matches.

BLAST, which stands for Basic Local Alignment Search Tool (Altschul, S.F. (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol.215:403-410), was used to search for local sequence alignments. BLASTproduces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially usefull in determining exact matches orin identifying homologs. BLAST is usefull for matches which do notcontain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labeledmicleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. 1993 and1990, supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding NANCH occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of NANCH-Encoding Polynucleotides to Full Length or toRecover Regulatory Sequences

Full length NANCH-encoding nucleic acid sequence (SEQ ID NO:2) is usedto design oligonucleotide primers for extending a partial nucleotidesequence to full length or for obtaining 5' or 3', intron or othercontrol sequences from genolic libraries. One primer is synthesized toinitiate extension in the antisense direction (XLR) and the other issynthesized to extend sequence in the sense direction (XLF). Primers areused to facilitate the extension of the known sequence "outward"generating amplicons containing new, unknown nucleotide sequence for theregion of interest. The initial primers ire designed from the cDNA usingOLIGO 4.06 (National Biosciences), or another appropriate program, to be22-30 nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68°-72° C. Anystretch of nucleotides which would result in hairpin structures andprimer-primer dimerizations is avoided.

The original, selected cDNA libraries, or a human genomic library areused to extend the sequence; the latter is most useful to obtain 5'upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) andthoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters:

    ______________________________________                                        Step 1       94° C. for 1 min (initial denaturation)                     Step 2 65° C. for 1 min                                                Step 3 68° C. for 6 min                                                Step 4 94° C. for 15 sec                                               Step 5 65° C. for 1 min                                                Step 6 68° C. for 7 min                                                Step 7 Repeat step 4-6 for 15 additional cycles                               Step 8 94° C. for 15 sec                                               Step 9 65° C. for 1 min                                                Step 10 68° C. for 7:15 min                                            Step 11 Repeat step 8-10 for 12 cycles                                        Step 12 72° C. for 8 min                                               Step 13  4° C. (and holding)                                         ______________________________________                                    

A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresison a low concentration (about 0.6-0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Bands thoughtto contain the largest products are selected and removed from the gel.Further purification involves using a commercial gel extraction methodsuch as QIAQuick™ (QIAGEN Inc. Chatsworth, Calif.). After recovery ofthe DNA, Klenow enzyme is used to trim single-stranded, nucleotideoverhangs creating blunt ends which facilitate relegation and cloning.

After ethanol precipitation, the products are redissolve in 13 μl ofligation buffer. 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2× Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2× Carbmedium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture is transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 μl of each sample istransferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

    ______________________________________                                        Step 1     94° C. for 60 sec                                             Step 2 94° C. for 20 sec                                               Step 3 55° C. for 30 sec                                               Step 4 72° C. for 90 sec                                               Step 5 Repeat steps 2-4 for an additional 29 cycles                           Step 6 72° C. for 180 sec                                              Step 7  4° C. (and holding)                                          ______________________________________                                    

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid, and sequenced.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³² P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonuclecotides are substantially purified with SephadexG-25 superfine resin column (Pharmacia & Upjohn). A portion containing10⁷ counts per minute of each of the sense and antisenseoligonucleotides is used in a typical membrane based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DuPontNEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1 × salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak. Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

VII Antisense Molecules

Antisense molecules to the NANCH-encoding sequence, or any part thereof,is used to inhibit in vivo or in vitro expression of naturally occurringNANCH. Although use of antisense oligonucleotides, comprising about 20base-pairs, is specifically described, essentially the same procedure isused with larger cDNA fragments. An oligonucleotide based on the codingsequences of NANCH, as shown in FIGS. 1A and 1B, is used to inhibitexpression of naturally occurring NANCH. The complementaryoligonucleotide is designed from the most unique 5' sequence as shown inFIGS. 1A and 1B and used either to inhibit transcription by preventingpromoter binding to the upstream nontranslated sequence or translationof an NANCH-encoding transcript by preventing the ribosome from binding.Using an appropriate portion of the signal and 5' sequence of SEQ IDNO:2, an effective antisense oligonucleotide includes any 15-20nucleotides spanning the region which translates into the signal or 5'coding sequence of the polypeptide as shown in FIGS. 1A and 1B.

VIII Expression of NANCH

Expression of NANCH is accomplished by sub cloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express NANCH in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofNANCH into the bacterial growth media which can be used directly in thefollowing assay for activity.

IX Demonstration of NANCH Activity

NANCH can be expressed by transforming a mammalian cell line such asCOS7, HeLa or CHO with an eukaryotic expression vector encoding NANCH.Eukaryotic expression vectors are commercially available, and thetechniques to introduce them into cells; are well known to those skilledin the art. A small amount of a second plasmid, which expresses any oneof a number of marker genes such as β-galactosidase, is co-transformedinto the cells in order to allow rapid identification of those cellswhich have taken up and expressed the foreign DNA. The cells areincubated for 48-72 hours after transformation under conditionsappropriate for the cell line to allow expression and accumulation ofNANCH and β-galactosidase.

Transformed cells expressing β-galactosidase are stained blue when asuitable colorimetric substrate is added to the culture media underconditions that are well known in the art. Stained cells are tested fordifferences in membrane conductance due to chloride ions or other anionsby electrophysiological techniques that are well known in the art.Untransformed cells, and/or cells transformed with either vectorsequences alone or β-galactosidase sequences alone, are used as controlsand tested in parallel.

Cells expressing NANCH will have higher anionic conductance than controlcells. The contribution of NANCH to the anionic conductance can beconfirmed by incubating the cells using antibodies specific for NANCH.The NANCH-specific antibodies will bind to the extracellular side ofNANCH and thereby block the sore in the ion channel.

X Production of NANCH Specific Antibodies

NANCH that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

XI Purification of Naturally Occurring NANCH Using Specific Antibodies

Naturally occurring or recombinant NANCH is substantially purified byimmunoaffinity chromatography using antibodies specific for NANCH. Animmunoaffinity column is constructed by covalently coupling NANCHantibody to an activated chromatographic resin, such as CnBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing NANCH is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof NANCH (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/NANCH binding (e.g., a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and NANCH iscollected.

XII Identification of Molecules Which Interact with NANCH

NANCH or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled NANCH, washed and any wells withlabeled NANCH complex are assayed. Data obtained using differentconcentrations of NANCH are used to calculate values, for the number,affinity, and association of NANCH with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 253 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: Consensus                                                        (B) CLONE: Consensus                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Met Ala Leu Ser Met Pro Leu Asn Gly Leu Ly - #s Glu Glu Asp Lys        Glu                                                                              1               5  - #                10  - #                15              - - Pro Leu Ile Glu Leu Phe Val Lys Ala Gly Se - #r Asp Gly Glu Ser Ile                  20      - #            25      - #            30                   - - Gly Asn Cys Pro Phe Ser Gln Arg Leu Phe Me - #t Ile Leu Trp Leu Lys              35          - #        40          - #        45                       - - Gly Val Val Phe Ser Val Thr Thr Val Asp Le - #u Lys Arg Lys Pro Ala          50              - #    55              - #    60                           - - Asp Leu Gln Asn Leu Ala Pro Gly Thr His Pr - #o Pro Phe Ile Thr Phe      65                  - #70                  - #75                  - #80        - - Asn Ser Glu Val Lys Thr Asp Val Asn Lys Il - #e Glu Glu Phe Leu Glu                      85  - #                90  - #                95               - - Glu Val Leu Cys Pro Pro Lys Tyr Leu Lys Le - #u Ser Pro Lys His Pro                  100      - #           105      - #           110                  - - Glu Ser Asn Thr Ala Gly Met Asp Ile Phe Al - #a Lys Phe Ser Ala Tyr              115          - #       120          - #       125                      - - Ile Lys Asn Ser Arg Pro Glu Ala Asn Glu Al - #a Leu Glu Arg Gly Leu          130              - #   135              - #   140                          - - Leu Lys Thr Leu Gln Lys Leu Asp Glu Tyr Le - #u Asn Ser Pro Leu Pro      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Glu Ile Asp Glu Asn Ser Met Glu Asp Il - #e Lys Phe Ser Thr        Arg                                                                                             165  - #               170  - #               175             - - Lys Phe Leu Asp Gly Asn Glu Met Thr Leu Al - #a Asp Cys Asn Leu Leu                  180      - #           185      - #           190                  - - Pro Lys Leu His Ile Val Lys Val Val Ala Ly - #s Lys Tyr Arg Asn Phe              195          - #       200          - #       205                      - - Asp Ile Ser Lys Glu Met Thr Gly Ile Trp Ar - #g Tyr Leu Thr Asn Ala          210              - #   215              - #   220                          - - Tyr Ser Arg Asp Gly Phe Thr Asn Thr Cys Pr - #o Ser Asp Lys Glu Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Glu Ile Ala Tyr Ser Asp Val Ala Lys Arg Le - #u Thr Lys                                  245  - #               250                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 819 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: Consensus                                                        (B) CLONE: Consensus                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GCAGCCGAGC CGGCCATGGC GTTGTCGATG CCGCTGAATG GGCTGAAGGA GG -            #AGGACAAA     60                                                                 - - GAGCCCCTCA TCGAGCTCTT CGTCAAGGCT GGCAGTGATG GTGAAAGCAT AG -            #GAAACTGC    120                                                                 - - CCCTTTTCCC AGAGGCTCTT CATGATTCTT TGGCTCAAAG GAGTTGTATT TA -            #GTGTGACG    180                                                                 - - ACTGTTGACC TGAAAAGGAA GCCAGCAGAC CTGCAGAACT TGGCTCCCGG GA -            #CCCACCCA    240                                                                 - - CCATTTATAA CTTTCAACAG TGAAGTCAAA ACGGATGTAA ATAAGATTGA GG -            #AATTTCTT    300                                                                 - - GAAGAAGTCT TATGCCCTCC CAAGTACTTA AAGCTTTCAC CAAAACACCC AG -            #AATCAAAT    360                                                                 - - ACTGCTGGAA TGGACATCTT TGCCAAATTC TCTGCATATA TCAAGAATTC AA -            #GGCCAGAG    420                                                                 - - GCTAATGAAG CACTGGAGAG GGGTCTCCTG AAAACCCTGC AGAAACTGGA TG -            #AATATCTG    480                                                                 - - AATTCTCCTC TCCCTGATGA AATTGATGAA AATAGTATGG AGGACATAAA GT -            #TTTCTACA    540                                                                 - - CGTAAATTTC TGGATGGCAA TGAAATGACA TTAGCTGATT GCAACCTGCT GC -            #CCAAACTG    600                                                                 - - CATATTGTCA AGGTGGTGGC CAAAAAATAT CGCAACTTTG ATATTTCAAA AG -            #AAATGACT    660                                                                 - - GGCATCTGGA GATACCTAAC TAATGCATAC AGTAGGGACG GGTTCACCAA TA -            #CCTGTCCC    720                                                                 - - AGTGATAAGG AGGTTGAAAT AGCATATAGT GATGTAGCCA AAAGACTCAC CA -            #AGTAAAAT    780                                                                 - - CGCGTTTGTA AAAGAGGTGT CTTCATGTCT TCCCCTAAG      - #                      - #   819                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 437 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 289404                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Met Asn Asp Glu Asn Tyr Ser Thr Thr Ile Ty - #r Asn Arg Val Gln Thr       1               5  - #                10  - #                15               - - Glu Arg Val Tyr Glu Asp Ser Asp Pro Ala Gl - #u Asn Gly Gly Pro Leu                  20      - #            25      - #            30                   - - Tyr Asp Glu Val His Glu Asp Val Arg Arg Gl - #u Asp Asn Leu Tyr Val              35          - #        40          - #        45                       - - Asn Glu Leu Glu Asn Gln Glu Tyr Asp Ser Va - #l Ala Val Tyr Pro Val          50              - #    55              - #    60                           - - Gly Arg Gln Gly Arg Thr Ser Ala Ser Leu Gl - #n Pro Glu Thr Gly Glu      65                  - #70                  - #75                  - #80        - - Tyr Val Leu Pro Asp Glu Pro Tyr Ser Lys Al - #a Gln Asp Pro His Pro                      85  - #                90  - #                95               - - Gly Glu Pro Thr Ala Asp Glu Asp Ile Ser Le - #u Glu Glu Leu Leu Ser                  100      - #           105      - #           110                  - - Pro Thr Lys Asp His Gln Ser Asp Ser Glu Gl - #u Pro Gln Ala Ser Asp              115          - #       120          - #       125                      - - Pro Glu Glu Pro Gln Ala Ser Asp Pro Glu Gl - #u Pro Gln Gly Pro Asp          130              - #   135              - #   140                          - - Pro Glu Glu Pro Gln Glu Asn Gly Asn Glu Me - #t Glu Ala Asp Leu Pro      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ser Pro Ser Ser Phe Thr Ile Gln Asn Ser Ar - #g Ala Phe Ser Thr        Arg                                                                                             165  - #               170  - #               175             - - Glu Ile Ser Pro Thr Ser Tyr Ser Ala Asp As - #p Val Ser Glu Gly Asn                  180      - #           185      - #           190                  - - Glu Ser Ala Ser Ala Ser Pro Glu Ile Asn Le - #u Phe Val Lys Ala Gly              195          - #       200          - #       205                      - - Ile Asp Gly Glu Ser Ile Gly Asn Cys Pro Ph - #e Ser Gln Arg Leu Phe          210              - #   215              - #   220                          - - Met Ile Leu Trp Leu Lys Gly Val Val Phe As - #n Val Thr Thr Val Asp      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Lys Arg Lys Pro Ala Asp Leu His Asn Le - #u Ala Pro Gly Thr        His                                                                                             245  - #               250  - #               255             - - Pro Pro Phe Leu Thr Phe Asn Gly Asp Val Ly - #s Thr Asp Val Asn Lys                  260      - #           265      - #           270                  - - Ile Glu Glu Phe Leu Glu Glu Thr Leu Thr Pr - #o Glu Lys Tyr Pro Arg              275          - #       280          - #       285                      - - Leu Ala Ala Lys His Arg Glu Ser Asn Thr Al - #a Gly Ile Asp Ile Phe          290              - #   295              - #   300                          - - Val Lys Phe Ser Ala Tyr Ile Lys Asn Thr Ly - #s Gln Gln Ser Asn Ala      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ala Leu Glu Arg Gly Leu Thr Lys Ala Leu Ly - #s Lys Leu Asp Asp        Tyr                                                                                             325  - #               330  - #               335             - - Leu Asn Thr Pro Leu Pro Glu Glu Ile Asp Al - #a Asp Thr Arg Gly Asp                  340      - #           345      - #           350                  - - Asp Glu Lys Gly Ser Arg Arg Lys Phe Leu As - #p Gly Asp Glu Leu Thr              355          - #       360          - #       365                      - - Leu Ala Asp Cys Asn Leu Leu Pro Lys Leu Hi - #s Val Val Lys Ile Val          370              - #   375              - #   380                          - - Ala Lys Lys Tyr Arg Asn Tyr Asp Phe Pro Al - #a Glu Met Thr Gly Leu      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Trp Arg Tyr Leu Lys Asn Ala Tyr Ala Arg As - #p Glu Phe Thr Asn        Thr                                                                                             405  - #               410  - #               415             - - Cys Ala Ala Asp Ser Glu Ile Glu Leu Ala Ty - #r Ala Asp Val Ala Lys                  420      - #           425      - #           430                  - - Arg Leu Ser Arg Ser                                                              435                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 210 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 895845                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Val Leu Trp Leu Lys Gly Val Thr Phe As - #n Val Thr Thr Val Asp       1               5  - #                10  - #                15               - - Thr Lys Arg Arg Thr Glu Thr Val Gln Lys Le - #u Cys Pro Gly Gly Gln                  20      - #            25      - #            30                   - - Leu Pro Phe Leu Leu Tyr Gly Thr Glu Val Hi - #s Thr Asp Thr Asn Lys              35          - #        40          - #        45                       - - Ile Glu Glu Phe Leu Glu Ala Val Leu Cys Pr - #o Pro Arg Tyr Pro Lys          50              - #    55              - #    60                           - - Leu Ala Ala Leu Asn Pro Glu Ser Asn Thr Al - #a Gly Leu Asp Ile Phe      65                  - #70                  - #75                  - #80        - - Ala Lys Phe Ser Ala Tyr Ile Lys Asn Ser As - #n Pro Ala Leu Asn Asp                      85  - #                90  - #                95               - - Asn Leu Glu Lys Gly Leu Leu Lys Ala Leu Ly - #s Val Leu Asp Asn Tyr                  100      - #           105      - #           110                  - - Leu Thr Ser Pro Leu Pro Glu Glu Val Asp Gl - #u Thr Ser Ala Glu Asp              115          - #       120          - #       125                      - - Glu Gly Val Ser Gln Arg Lys Phe Leu Asp Gl - #y Asn Glu Leu Thr Leu          130              - #   135              - #   140                          - - Ala Asp Cys Asn Leu Leu Pro Lys Leu His Il - #e Val Gln Val Val Cys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Lys Tyr Arg Gly Phe Thr Ile Pro Glu Al - #a Phe Arg Gly Val        His                                                                                             165  - #               170  - #               175             - - Arg Tyr Leu Ser Asn Ala Tyr Ala Arg Glu Gl - #u Phe Ala Ser Thr Cys                  180      - #           185      - #           190                  - - Pro Asp Asp Glu Glu Ile Glu Leu Ala Tyr Gl - #u Gln Val Ala Lys Ala              195          - #       200          - #       205                      - - Leu Lys                                                                      210                                                                      __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide sequenceencoding a human anion channel protein comprising the amino acidsequence of SEQ ID NO:1.
 2. An isolated and purified polynucleotidesequence which is completely complementary to the polynucleotidesequence of claim
 1. 3. A hybridization probe comprising thepolynucleotide sequence of claim 1 and a detectable label.
 4. Anisolated and purified polynucleotide sequence comprising SEQ ID NO:2. 5.An isolated and purified polynucleotide sequence which is completelycomplementary to the polynucleotide sequence of claim
 4. 6. Ahybridization probe comprising the polynucleotide sequence of claim 5and a detectable label.
 7. An expression vector containing thepolynucleotide sequence of claim
 1. 8. A host cell containing the vectorof claim
 7. 9. A method for producing a polypeptide comprising the aminoacid sequence of SEQ ID NO:1 the method comprising the steps of:a)culturing the host cell of claim 8 under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fronithe host cell culture.